A new approach for optimal morphological identification and immunolabeling of spermatogonial cells.

High quality fixation often inactivates epitopes and gentler fixation can fail to preserve biological structure at the required resolution. For studies of male reproduction, immunofluorescence techniques using paraformaldehyde fixation associated with paraffin as an embedding medium gives good epitope preservation, although the cell becomes morphologically compromised. On the other hand, glutaraldehyde associated with a plastic resin has been used with success to recognize and distinguish each spermatogonial cell subtype, but the antigenic sites become inaccessible to antibodies. Here we describe a new method that provides excellent morphological details of testicular cells while preserving the binding capacity of epitopes. Using a combination of glutaraldehyde and paraformaldehyde as a fixative and LR White resin for embedding, we show that it is possible to clearly recognize spermatogonial subtypes (Aund, A-A4, In and B spermatogonia) on 1-µm thick-sections and to label epitopes such as bromodeoxyuridine, a marker used for cellular cycle studies in the testis. The information gained from this procedure can be critical for understanding spermatogonial process of self-renewal and differentiation.

Spermatogonial morphology and kinetics during testis development in mice: a high-resolution light microscopy approach.

Despite the knowledge of spermatogonial biology in adult mice, spermatogonial development in immature animals has not been fully characterized. Thus, the aim of this study was to evaluate the ontogeny of the morphological development of the spermatogonial lineage in C57BL/6 mouse testis, using high-resolution light microscopy. Spermatogonial morphology, chronology, and absolute number were determined for different ages postpartum (pp). The morphology of spermatogonia in immature mice was similar to that of adult spermatogonia, although their nuclear diameter was slightly smaller. The A(1) spermatogonia were first observed on day 2 pp, and only 24 h later, differentiating type A(3) and A(4) spermatogonia were observed in the seminiferous cords. This result indicated a shortening of the spermatogonial phase for immature mice of about ∼2.5 days when compared with adult mice and suggests that gonocytes and/or A(1) spermatogonia could directly become A(4) spermatogonia, skipping the developmental sequence of type A spermatogonia. These A(4) spermatogonia are functional as they develop into type B spermatogonia by day 5 pp. At day 8 pp, while differentiation to spermatocytes begins, the A(und) spermatogonia reach their maximal numbers, which are maintained through adulthood. The various details of the spermatogonial behavior in immature normal mice described in this study can be used as a baseline for further studies under experimental or pathological conditions.

Fetal cyclophosphamide exposure induces testicular cancer and reduced spermatogenesis and ovarian follicle numbers in mice.

Exposure to radiation during fetal development induces testicular germ cell tumors (TGCT) and reduces spermatogenesis in mice. However, whether DNA damaging chemotherapeutic agents elicit these effects in mice remains unclear. Among such agents, cyclophosphamide (CP) is currently used to treat breast cancer in pregnant women, and the effects of fetal exposure to this drug manifested in the offspring must be better understood to offer such patients suitable counseling. The present study was designed to determine whether fetal exposure to CP induces testicular cancer and/or gonadal toxicity in 129 and in 129.MOLF congenic (L1) mice. Exposure to CP on embryonic days 10.5 and 11.5 dramatically increased TGCT incidence to 28% in offspring of 129 mice (control value, 2%) and to 80% in the male offspring of L1 (control value 33%). These increases are similar to those observed in both lines of mice by radiation. In utero exposure to CP also significantly reduced testis weights at 4 weeks of age to ∼ 70% of control and induced atrophic seminiferous tubules in ∼ 30% of the testes. When the in utero CP-exposed 129 mice reached adulthood, there were significant reductions in testicular and epididymal sperm counts to 62% and 70%, respectively, of controls. In female offspring, CP caused the loss of 77% of primordial follicles and increased follicle growth activation. The results indicate that i) DNA damage is a common mechanism leading to induction of testicular cancer, ii) increased induction of testis cancer by external agents is proportional to the spontaneous incidence due to inherent genetic susceptibility, and iii) children exposed to radiation or DNA damaging chemotherapeutic agents in utero may have increased risks of developing testis cancer and having reduced spermatogenic potential or diminished reproductive lifespan.

Effects of multiple doses of cyclophosphamide on mouse testes: accessing the germ cells lost, and the functional damage of stem cells.

Spermatogenesis is sensitive to the chemotherapeutic drug cyclophosphamide, which decreases the patients' sperm count. Since the recovery of fertility is dependent on regeneration from stem cells, in the present study we evaluated the ability of cyclophosphamide-exposed stem spermatogonia from mice to regenerate spermatogenesis in situ and after transplantation. When seven doses of cyclophosphamide were given at 4-day intervals, the differentiating germ cells were largely eliminated but ~50% of the undifferentiated type A spermatogonia remained. We monitored the recovery and found that sperm production recovered to 64% of control within the time expected. When the cyclophosphamide-surviving spermatogonia were transplanted into recipient mice, recovery of spermatogenesis from the cyclophosphamide-exposed donor cells was observed, but was reduced when compared to cells from cryptorchid donors. Thus, multidose regimens of cyclophosphamide did not eliminate the stem spermatogonia, but resulted in cell loss and residual damage.